Briarane Diterpenes from the South China Sea Gorgonian Coral, Junceella gemmacea

Four new briarane diterpenoids, junceellolides M–P (1–4), were isolated together with seven known analogs (5–11) from the South China Sea gorgonian, Junceella gemmacea. The structures of these compounds were elucidated by detailed spectroscopic analysis and comparison with the reported data. The absolute configuration of compounds 1–3 were determined based on an ECD experiment, while the absolute configuration of compound 4 was genetically determined. All the compounds were isolated for the first time from J. gemmacea. These compounds showed no growth inhibitory activity against A549, MG63 and SMMC-7721 cell lines in an in vitro bioassay.


Introduction
Gorgonian corals of the genus Junceella (phylum, Cnidaria; class, Anthozoa; order, Gorgonacea; family, Ellisellidae) are widely distributed in the subtropical and tropical waters of the Indo-Pacific Ocean as whip-shaped unbranched colonies of variable colors. These animals are well-known as a source of highly oxidized diterpenes of the briarane class [1,2]. Briarane-related natural products are characterized by the presence of a γ-lactone fused to a bicyclo [8.4.0] ring system [3] and have attracted great attention due to their chemical diversity and wide spectrum of bioactivities, including cytotoxic [4], anti-inflammatory [5], antibacterial [6], immunomodulatory [7], anti-fouling [8] and insecticidal [9] effects. Chemical investigation on the genus of Junceella can be traced back to 1983, at which time the isolation of junceellin [10] was reported from the Chinese gorgonian Junceella squamata [1]. Since then, more than 140 briarane diterpenes have been isolated from members of this genus, which involves four species, namely J. juncea, J. fragilis, J. gemmacea and J. squamata [1,11]. Previous research activities have been focused on J. juncea and J. fragilis, and only a few reports have dealt with corals belonging to the species J. gemmacea and J. squamata [2].
In our ongoing search for novel and bioactive secondary metabolites from marine invertebrates of the South China Sea, we were attracted by the potential medicinal value of the briarane diterpenoids. Previously, we reported the isolation of a series of briarane-type diterpenes with antimicrobial and tumor cell growth inhibition activities from the gorgonian, Dichotella gemmacea [12][13][14][15]. These results inspired us to continue to examine this class of metabolites, leading to the present investigation of the gorgonian J. gemmacea which was previously reported to be a rich source of briarane diterpenes. Chemical investigation on the acetone extract of these animals resulted in the isolation of four new briarane diterpenes, junceellolides M-P (1-4), together with seven known ones, namely junceellolide A (5), junceellin A (6), praelolide (7), juncin ZI (8), junceellolide B (9), junceellolide C (10) and junceellolide D (11) (Figure 1). The structures of these compounds were elucidated by extensive spectroscopic analysis ( 1 H and 13 C NMR, DEPT, HSQC, HMBC, NOESY, 1 H-1 H COSY and HRESIMS) and comparison with the reported analytical data for the known compounds. We herein report the isolation, structural determination and bioactivities of these new compounds.

Results and Discussion
Colonies of the gorgonian coral J. gemmacea (Valenciennes) were immediately frozen after collection and stored at −20 °C until extraction. The frozen organism was cut into small pieces and extracted exhaustively with acetone and methanol at room temperature. The Et 2 O-soluble portion (13.0 g) of the acetone extract was chromatographed repeatedly over silica gel, Sephadex LH-20, and semi-preparative RP-HPLC, to afford compounds 1-11. By extensive spectroscopic analysis combined with careful comparison with the reported data, the structures of the known compounds were determined as junceellolide A (5) [16], junceellin A (6) [1,10], praelolide (7) [16], juncin ZI (8) [17], junceellolide B (9) [16], junceellolide C (10) [16] and junceellolide D (11) [16]. Junceellin A (6) and juncin ZI (8) were once reported from J. squamata and J. juncea, respectively, and their structures were elucidated on the basis of ESIMS, UV, IR and NMR techniques. The structures of junceellolides A-D (5, 9-11) and praelolide (7), previously obtained from the South China Sea gorgonian, J. fragilis, were also established by extensive spectroscopic analysis. Junceellolide M (1) was isolated as a white amorphous powder and had a molecular formula of C 28 H 36 O 12 deduced from HRESIMS, indicating eleven degrees of unsaturation. The IR spectrum of 1 showed absorption bands for γ-lactone (1781 cm −1 ) and ester carbonyl (1736 cm −1 ) groups. This observation was consistent with the signals in the 13 C NMR and DEPT spectra (Table 1) for nine sp 2 carbon atoms (5 × OC=O, CH=CH, CH=C) at lower field and nineteen sp 3 carbon atoms at higher field (1 × C, 2 × CH, 2 × CH 2 , 6 × CH 3 , 2 × OC, 4 × OCH, 2 × OCH 2 ), accounting for seven double-bond equivalents. The remaining double bond equivalents were due to the presence of three rings in the molecule.
The NMR data for compound 1 were almost identical to those of frajunolide D (12) [18], an analogue isolated from the gorgonian J. fragilis, except for the absence of one acetate ester group. Location of the missing acetate ester group to C-12 was indicated by the distinct correlations from H 2 -12 to H 2 -13 and to H-14 in a 1 H-1 H COSY experiment of 1 ( Figure 2). The planar structure of 1 was further supported by 1 H-1 H COSY and HMBC data, as shown in Figure 2.  The relative configuration of 1 was proven to be the same as that of frajunolide D (12) by a NOESY experiment (Figure 3), in which we have arbitrarily chosen the β-orientation of H-13β, H-14, Me-15, H-17 and H 2 -20 and an α-orientation of H-2, H-9, H-10, Me-18 and 8-OH. The geometry of the Δ 3 double bond was assigned as Z based on the proton coupling constant between H-3 and H-4 (J = 8.7 Hz), while the geometry of the Δ 5 double bond was E as deduced from an NOE correlation between H-6 and H-16a. The chemical shift value of C-11 and C-20 (δ C 60.1 and 50.5, respectively) supported the α-configurational assignment of the 11,20-epoxy group. It has been reported that the 13 C NMR shifts for C-11 and C-20 are at δ 62-63 and 58-60 when the epoxy group is β-oriented and at δ 55-61 and 47-52 ppm when α-oriented [19]. The relative configuration of 1 was thus determined as (1R*,2S*,3Z,5E,7S*,8S*,9S*,10S*,11R*,14S*,17R*).  Junceellolide N (2) was obtained as a white powder. The molecular formula was determined to be C 26 H 33 O 10 Cl by HRESIMS. An isotopic ratio of 3:1 was observed in the molecular ion peaks at m/z 563.1663/565.1782 [M + Na] + , further indicating the presence of one chlorine atom. The comparison of the overall 1 H and 13 C NMR data of 2 with those of 1 (Table 1) revealed a close similarity, except that the acetyl group at C-16 in 1 was replaced by a chlorine atom in 2. The structure was fully supported by the 2D NMR experiments, including 1 H-1 H COSY, HMBC and NOESY. Compound 2 also gave a similar negative ECD band at 202 nm and a positive one below 200 nm (Figure 4), and thus its absolute configuration was then determined as (−)- (1S,2S,3Z,5E,7S,8S,9S,10S,11R,14S,17R).
Junceellolide O (3) had a molecular formula of C 27 H 36 O 11 , as deduced from the HRESIMS. The 1 H and 13 C NMR data of 3 also closely resembled those of 1, except for the replacement of the acetyl group at C-16 in 1 by a methoxy group (δ H 3.43, δ C 58.5) in 3. This assignment was further confirmed by the distinct HMBC correlation from H 2 -16 to the methoxy carbon (δ C 58.5). The relative and absolute configuration of 3 was proven to be the same as that of 1 by analysis of the NOESY and ECD spectra, and was thus determined as (+)-(1S,2S,3Z,5E,7S,8S,9S,10S,11R,14S,17R).
The gross structure of compound 4 was characterized by a detailed analysis of 2D NMR spectra. to C-1, C-2, C-10 and C-14, H 2 -16 to C-4 and C-5, H-6 to C-16, H-17 to C-7, C-8, C-9 and C-19, H-9 to C-8 and H 2 -20 to C-10, C-11 and C-12, respectively ( Figure 5). The relative configurations at chiral centers were proven to be the same as those of compounds 1-3. Interestingly, the large coupling constant for the olefinic protons (J = 16.8 Hz) indicated an E geometry of Δ 3 in 4 in contrast to those of the Z double bonds in structures 1-3. To our knowledge, this is the first report of a trans Δ 3,5 conjugated diene in a briarane diterpenoid. The relative configuration of 4 was established by NOE ( Figure 6) and coupling constant analysis whereas we propose its absolute configuration as (1R,2S,3E,5E,7S,8S,9S,10S,11R,14S,17R)-4, due to its biogenetic correlation with those of compounds 1-3.
Compounds 1-11 were evaluated for their ability to inhibit tumor cell growth using the A549, MG63 and SMMC-7721 cell lines [20]. None of these compounds exhibited growth inhibitory effects against these cell lines in the in vitro bioscreening (IC 50 ≥ 40 µM).

General Experimental Procedures
Commercial silica gel (Yantai, China, 200-300; 400-500 mesh) and RP silica gel (Merck, Darmstadt, Germany, 43-60 µm) were used for column chromatography (CC). Sephadex LH-20 (GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden) was used for either purification or separation. TLC was carried out on precoated silica gel plates (Yantai, China, HSGF-254) and RP silica gel (Macherey-Nagel, Düren, Germany, RP-18 F254), and spots were detected on TLC under UV and visualized by spraying with anisaldehyde-sulfuric acid reagent, followed by heating. HPLC was performed using a system comprised of an Agilent G1311A pump, an Agilent G1315B DAD detector and a Rheodyne 7725 injection port. A semi-preparative normal-phase column (YMC Pack ODS-A, 250  10 mm I.D., particle size 5 µm, 250 × 10 mm, YMC Europe GmbH, Dinslaken, Germany) was used for HPLC. The NMR data were recorded on a Bruker DRX 400 and Avance 500 spectrometers at 400 or 500 MHz for 1 H and 100 or 125 MHz for 13 C, respectively. Chemical shifts are reported in parts per million (δ), with the use of the residual CDCl 3 signal (δ H = 7.27 ppm) as an internal standard for 1 H NMR and CDCl 3 (δ C = 77.02 ppm) for 13 C NMR, and the coupling constants (J) were in Hz. The 1 H and 13 C NMR assignments were supported by 1 H-1 H COSY, HSQC, HMBC and NOESY experiments. The following abbreviations are used to describe spin multiplicity: s denotes singlet; d denotes doublet; t denotes triplet; m denotes multiplet; br s denotes broad singlet; br d denotes broad doublet; dd denotes doublet of doublets; tt denotes triplet of triplets; ov denotes overlapped signals. Optical rotations were measured in CH 2 Cl 2 with an Autopol IV polarimeter at the sodium D line (590 nm). Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet, USA); peaks are reported in cm −1 . UV absorption spectra were recorded on a Varian Cary 100 UV-Vis spectrophotometer; peak wavelengths are reported in nanometers. Circular dichroism spectra were recorded with a JASCO J-715 circular dichroism spectropolarimeter. The HRMS were performed on a Q-TOF micro-mass spectrometer (resolution: 5000). An isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound.

Animal Material
Specimens of the gorgonian coral Junceella gemmacea were collected from the South China Sea in October 2011, and identified by Xiu-Bao Li of the South China Sea Institute of Oceanology, Academia Sinica. The voucher specimen was deposited in the Second Military Medical University, Shanghai, China.
The isolation of an array of briarane diterpenoids demonstrates the productive chemical diversity of the gorgonian coral J. gemmacea, similar to that found in other species of this genus as well as in the genus Dichotella [2,20,22] The complexity of these intriguing structures and variable potential for tumor cell growth inhibition activity of compounds in this structure class may attract additional attention from chemists and pharmacologists, and encourage further investigations on the chemistry and antitumor activity of this cluster of metabolites.